The present application represents the next phase of a 20 year program in which the long term objective is the integration of subcellular organelle Ca compartmentation, Ca movements and contractile state in the intact functional mammalian cardiac cell to provide a more complete model for the cardiac excitation-contraction coupling (ECC) sequence. The specific aims of the proposal are: (1) Correlation of organelle Ca content, organelle flux, cytosolic free Ca transient, Ca currents and contractile response for a variety of physiological and pharmacological interventions in order to develop a more complete model for the cardiac ECC sequence. Apply the mathematical modeling technique developed by us to provide additional insight into the interaction among organelles/compartments with respect to subcellular Ca movement in the different inotropic states. (2) Correlation of the cellular adenine nucleotide level with alterations in SL phospholipid, cellular Ca compartmentation and contraction in order to better define the cellular response to energy deprivation. Particular attention will be given to the sequence of alteration SL Ca permeability, SL phospholipid asymmetry change and more severe SL alteration leading to cell lysis at various energy levels. These aims will e approached using the following methods (all presently operative in the Laboratory) (1) Isolated adult cell and tissue culture techniques; (2) Video analysis of cell contraction under conditions of rapid superfusion; (3) whole cell and patch-clamp techniques; (4) Non-perfusion limited 45Ca exchange techniques for labeling and washout of adult cells and cultured cells based on unique scintillation technology developed by the Laboratory; (5) Epifluorescence monitoring of cellular Ca under conditions of rapid perfusion. Determination of free [Ca]i levels to be compared with state of subcellular Ca compartmentation; (6) SL isolation by the unique 'gas- dissection' technique developed by the Laboratory; (7) All standard techniques for lipid, protein and sugar analysis and for analysis of transport functions (Ca pump, Na-Ca exchange, Na-K ATPase). The manner by which the cardiac cell handles its Ca plays a major role in the determination of its level of contraction under physiological conditions. The design of inotropic agents and of interventions to prolong myocardial viability in the face of ischemia/reperfusion will be aided by further knowledge of subcellular Ca metabolism. The goal of this application is to provide this knowledge.